The proposed research concerns the in vitro evolution of novel nucleic acid enzymes that can be used to modulate biological processes. Nucleic adds have both genetic and catalytic properties, making it straightforward to couple amplification and mutation of their genetic sequence with selection based on their corresponding catalytic properties. Efforts will focus on the development of two classes of catalytic nucleic acids: DNA enzymes with N-glycosylase activity and RNA enzymes with homing endoribonudease activity. The former will be evolved to repair specific lesions of DNA that result from mutation or oxidative damage. The latter will be constructed by joining two existing RNA enzymes to create a bifunctional molecule that can insert itself in an irreversible manner at a specific location within RNA. It will be used to perform targeted gene disruption or targeted insertion of a coding region within mRNA. Attention also will be directed toward understanding the evolution process itself. Comparisons will be made among molecules that arise as a consequence of different degrees of selection pressure or varying complexity of their component subunits. A new approach employing a quench-flow device will be used to evolve ENA enzymes with very fast reaction rates. RNA enzymes also will be developed that operate under conditions of extreme pH or temperature, shedding light on the limits of RNA-based catalytic function. Finally, a new class of tethered small-molecule cofactors will be synthesized and supplied to the nucleic add enzymes to assist in their catalytic function. These cofactors will consist of either an amino add or a short peptide that is attached to the end of an oligodeoxynucleotide adapter. The adapter will allow the cofactor to be bound readily by the enzyme, allowing evolution to exploit the bound cofactor for use in catalysis.